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Pilot Program Grants Program (NBIC), 2013

IN-SITU ELECTRON MICROSCOPY FOR NANO-SCALE PROBING

A top view photo and a cross-section graphic of the nanoaquarium – the UPenn liquid cell for electron microscope imaging of objects suspended in and processes taking place in liquid media. The flow cell is about 100nm tall and sandwiched between two 50nm thick silicon nitride membranes. The device is transparent to electrons.

Electron microscopy with its nanoscale resolution has led to numerous important discoveries across diverse disciplines, ranging from material science to biology to medicine. Traditional electron microscopy is, however, limited to static, dry samples. In recent years, there has been a growing interest in enabling electron microscopy of dynamic processes in liquid media, under extreme conditions, and of specimens subjected to stresses. This emerging field is broadly defined as in-situ electron microscopy. In this pilot grant, our main objective is to develop methodology for and demonstrate imaging of macromolecules in liquids. As a model system, we will examine the interactions between the processive molecular motor myosin V and actin filaments. If successful, this technology could represent a major shift in how to study the dynamics of and interactions among biological macromolecules, with broad applications across many biomedical fields. The nanoaquarium is also useful for the study of directed and undirected assembly of colloidal crystals, crystallization processes, etching, electroplating and thin film morphology, electrochemical processes, nucleation, nanobubble stability, and radiolysis.

DEVELOPMENT OF A DIRECT IN-SITU PROBE OF ELECTROCHEMICAL PROCESSES WITH NM SPATIAL RESOLUTION FOR THE NBIC LOCAL PROBE FACILITY

1a. Diagram of the operation of a solid oxide fuel cell. b. Illustration of high temperature environmental cell for SPM.

In systems ranging from fuel cells to artificial photosynthesis to chemical catalysis, mass and charge transport occur in reactive, high temperature and dynamic environments. The ability to probe local interactions in realistic environments is prerequisite to understanding the behavior of these systems. Here a new environmental chamber for Scanning Probe Microscopy enables electronic and chemical processes to be investigated at temperatures as high at 1000°C, in the presence of reactive gases, with nm spatial resolution. This is being used to determine the mechanisms of charge and energy transfer at triple phase boundaries and ‘active zones’ in a solid oxide fuel cell. This represents a first look at these processes in situ during eletrochemical reactions.

3D images of fibroblast cells (diagonal left to right) showing the height (left) and elastic modulus (right). Note that stiffness increases as one moves down the topography gradient towards the cell periphery. Because probe depth is only 10nm, stiffness changes are likely due to cytoskeletal heterogeneity.

Robert Carpick
Department of Mechanical Engineering and Applied Mechanics

Cell stiffness underlies health issues from cancer and obesity to toxicology, and regenerative medicine. This pilot aims to provide a better fundamental understanding of diseases by investigating how environmental cues affect cell viscoelasticity. At present, it remains unclear how cell stiffness correlates to cell health, or if a spatial heterogeneity of stiffness across the cell can serve as a marker for overall cell health. Current microscopy techniques are unable to map cell viscoelasticity while simultaneously imaging subcellular components with organelle level resolution. The ability to simultaneously quantify cell mechanics and track fluorescent markers that reflect cell health at single and multiple cell level will enable us to correlate cell viscoelasticity with biophysical changes. Expanding the impact of NBIC facilities, Aim 1 develops AFM/TIRF and quartz-crystal microbalance with dissipation (QCM-D) methods to quantify how environmental cues alter cellular mechanical integrity and in turn relate cell mechanics to cell health. This study will require new methods to analyze the nonlinear viscoelastic nanoscale contact mechanics of cells. Using these methods, Aim 2 investigates viscoelastic mapping of endothelial cells, fibroblasts, and macrophages exposed to nanoparticles (NPs) and/or attached to dextran hydrogels with increasing stiffness.

INTEGRATED MOLECULAR DETECTION (iMD) CHIP

The integrated molecular detection (iMD) chip for ultra-sensitive profiling of sparse molecules in drinking water. a.The targeted molecules are captured onto magnetic nanoparticles (MNPs) using engineered affinity ligands. The microfluidic chip efficiently mixes the MNPs with the sample using chaotic mixing, efficiently utilizing valuable reagents. b. The magnetic beads are then isolated from the unprocessed sample using the track etched magnetic micropore filter (TEMPO). c.The enriched concentration of opioids is then released, using integrated Joule heating, and the lysate is delivered to an array of graphene biosensors for molecular profiling.

Nano-biosensors can detect molecular biomarkers with extremely high sensitivity and have been successfully developed to probe a wide range of biological and chemical targets. However, resolving sparse molecules in large volume samples, such as drinking water that contain vast amounts of other cells and molecules, remains challenging. Consequently, extensive sample preparation and purification are necessary before analysis, often leading to the decay of molecular biomarkers and the complication of use in remote settings. To overcome these challenges, we are developing an integrated molecular detection (iMD) chip. This chip uses a novel micro-magnetic sorting strategy to pre-enrich for a specific population of small molecules. The enriched sample is then analyzed by a downstream array of chemically functionalized graphene biosensors. The iMD platform will be self-contained, enabling it to function as a portable laboratory even in resource-limited, remote settings.

A) Cyclic voltammograms for purely polymer device at different scan rates. B) Cyclic voltammograms for a hybrid device at different scan rates C) Capacitance as a function of scan rate for different devices.

We are pursuing the understanding of polymer based electrochemical capacitors (pseudo capacitors) using for the redox electrode the conductive polymer, poly-3,4-propylenedioxythiophene (p-ProDOT) and p-ProDOT device as a bench mark. We are working on hybrids capacitors, which combines the redox driven mechanism for energy storage of pseudo- capacitors with the electrochemical double layer (EDL) physical phenomena. The first hybrid studied involved polymer-wrapped CNTS. We would like to enhance the energy density of the redox part of the device, while maintaining the power density characteristic of the EDL electrode. The synthetic methods utilized in the production of the precursors, and the lifetime of the device, while taking into consideration the potential hazards of its final disposal, costs of production and disposal, environmental impact, and processing to scale-up is being systematically explored. Other carbon materials (AC, CDC and CNO) that potentially can meet these demands without sacrificing performance are being explored. The effect of porosity at the nano-scale as well as the best methods in composites fabrication is currently under study.

Schematic of hydrodynamic confined microfluidic probe. A local microfluidic flow is created beneath the probe, which is positioned above a surface in a liquid-filled well. Top: Side view showing the positioning of the probe in a well on an inverted microscope. Bottom: Plan view of confined flow that is generated beneath the probe.

Over the past two decades, microfluidic devices have enabled unprecedented control of biological environments for studying cells, proteins, and membranes. Despite, the significant advantages and applications of microfluidics, the closed-channel geometry of traditional microfluidics limits there use in certain biological and medical research applications. Recently, a new strategy for generating microfluidic flows in open liquid environments using a hydrodynamic confined microfluidic (HCM) probes has been demonstrated. The objective of this pilot project is to develop HCM probes for sub-micrometer patterning of biomolecules in liquid environments and devices for single-cell electroporation.